Within this application several publications are referenced by Arabic numerals within parentheses. Full citations for these references may be found at the end of the specification immediately preceding the claims. The disclosures of these publications in their entirety are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
The subject of this invention is a vaccine for pseudo-rabies virus (herpesvirus suis, suid herpesvirus 1, or Aujesky's disease virus) disease of swine. Swine are the natural host of pseudorabies virus in which infection in older animals is commonly inapparent but may be characterized by fever, convulsions, and death particularly in younger animals. Pseudorabies also infects cattle, sheep, dogs, cats, ferrets, foxes, and rats (1) where the infection usually results in death. Death is usually preceded by intense pruritus, mania, encephalitis, paralysis, and coma. Traditional live vaccines are available for use in swine, but they are lethal for the other animals. An improved vaccine for pseudorabies would induce a more reliable immune response in swine, would be specifically attenuated to be incapable of reversion to virulence, and would not cause disease in other hosts.
In addition to the attributes given above, it would be advantageous to deliver in a single injection with the pseudorabies vaccine other antigens protective against other economically important diseases, for example rotavirus, transmissible gastroenteritis virus, and parvovirus. Such a vaccine would minimize the handling of the animal and the cost of administration. If these antigens were incorporated into the pseudorabies virus as part of the genome of the virus and were designed to be expressed during vaccine virus replication, other advantages would be realized. First, the cost of producing such vaccines would be lowered because the other antigens would be included in the cost of producing a single dose of pseudorabies vaccine. Second, the antigens cloned into pseudorabies virus would be safe --they could never revert to virulence because the majority of the virus nucleic acid sequences are not present in the vaccine. Thirdly, the vaccines would be delivered via a live virus vector that would replicate in living cells in the body which would promote the best possible immune response to give solid protection over the longest time period.
The present invention concerns pseudorabies viruses which have been genetically engineered to contain foreign DNA sequences which encode anitigens which are antigenic in the host animal and may be used to elicit protection against a number of different diseases. These viruses comprise a portion of the pseudorabies virus DNA which is essential for replication of the naturally-occurring virus, but which is missing DNA sequences that are required for full pathogenicity (i.e. they have attenuating deletions). Into these attenuated pseudorabies viruses have been inserted foreign genes under the control of various herpesvirus promoters that direct the expression of foreign antigenic proteins. Vaccines comprised of these viruses are characterized by the efficacy, safety, and economic benefits noted above.
The prior art for this invention stems first from the ability to clone and analyze DNA while in bacterial plasmids. The techniques that are available for the most part are detailed in Maniatis et al. (3112). This publication teaches state of the art general recombinant DNA techniques.
Among the herpesviruses, only two primate herpesviruses (herpes simplex of humans and, to a limited extent, herpes saimiri of monkeys) have been engineered to contain foreign DNA sequences previous to this disclosure. The earliest work on the genetic manipulation of herpes simplex involved the rescue of temperature sensitive mutants of the virus using purified restriction fragments of DNA (3). This work did not involve cloning of the DNA fragments nor the purposeful creation of deletions nor insertions of foreign DNA fragments into the viral genome. The first use of recombinant DNA to manipulate herpes simplex virus involved cloning a piece of DNA from the L-S junction region into the unique long region of the DNA, specifically into the thymidine kinase gene (4). This insert was not a foreign piece of DNA, rather it was a naturally-occurring piece of herpesvirus DNA that was duplicated at another place in the genome. This piece of DNA was not engineered to specifically express any protein, and thus it did not teach how to express protein in herpesviruses. The manipulation of herpes simplex next involved the creation of deletions in the virus genome by a combination of recombinant DNA and thymidine kinase selection. The first step was to make a specific deletion of the thymidine kinase gene (5). The next step involved the insertion of the thymidine kinase gene into the genome at a specific site, and then the thymidine kinase gene and the flanking DNA at the new site were deleted by a selection against thymidine kinase (6). In this manner herpes simplex alpha-22 gene has been deleted (6). In the most recent refinement of this technique, a 15,000 bp sequence of DNA has been deleted from the internal repeat of herpes simplex virus (7).
The insertion of genes that encode protein into primate herpesviruses have involved seven cases: the insertion of herpes simplex glycoprotein C back into a naturally occurring deletion mutant of this gene in herpes simplex virus (8); the insertion of glycoprotein D of herpes simplex type 2 into herpes simplex type 1 (9), again with no manipulation of promoters since the gene is not really `foreign`; the insertion of hepatitis B surface antigen into herpes simplex virus under the control of the herpes simplex ICP4 promoter (10); and the insertion of bovine growth hormone into herpes saimiri virus with an SV40 promoter that in fact didn't work in that system (an endogenous upstream promoter served to transcribe the gene) (11). Two additional cases of foreign genes (chicken ovalbumin gene and Epstein-Barr virus nuclear antigen) have been inserted into herpes simplex virus (18), and glycoprotein X of pseudorabies virus has been inserted into herpes simplex virus (20).
These limited cases of deletion and insertion of genes into primate herpesviruses demonstrate that it is possible to genetically engineer primate herpesvirus genomes by recombinant DNA techniques. The methods that have been used to insert genes involve homologous recombination between the viral DNA cloned on plasmids and purified viral DNA transfected into the same animal cell. In aggregate this is referred to as the homologous recombination technique. This technique has been adapted with some modifications to allow us to engineer pseudorabies virus. Several key elements of the genetic engineering of pseudorabies are not made obvious from these previous primate herpesvirus studies. The present invention demonstrates where to make deletions that serve to attenuate pseudorabies virus, where to make the insertions of the foreign genes to get them stably contained within the pseudorabies virus genome, and which promoters are effective in expressing foreign proteins in the pseudorabies virus genome.
Pseudorabies virus is classified as an alphaherpesvirus with a class D genome structure (12); that is, it contains two copies of a single repeat region, one located between the unique long and unique short DNA region and one at the terminus of the unique short region (see FIG. 1). Herpes simplex virus is an alphaherpesvirus with a class E genome (12); that is , it contains two copies of each of two repeats. Herpes saimiri is a gammaherpesvirus with a class B genome: that is, it contains numerous reiterations of the same sequence at both termini (12). As the genome structure differs significantly between these different classes of herpesviruses, and because the different viruses attack different cells within their hosts and elicit different pathologies, it is necessary in each instance to establish which specific regions can be removed in order to attenuate and which regions can be altered to express foreign genes.
Pseudorabies virus has been studied using the tools of molecular biology including the use of recombinant DNA techniques. BamHI, KpnI, and BglII restriction maps of the virus genome have been published (13, 14). DNA transfection procedures have been utilized to rescue temperature sensitive and deletion mutants of the virus by the homologous recombination procedure (13). There are two examples of deletions that have been made in the pseudorabies virus genome --one is a thymidine kinase gene deletion (15, 19) disclosed in U.S. Pat. No. 4,514,497 entitled "Modified Live Pseudorabies Viruses". This patent describes a method to delete the thymidine kinase gene of pseudorabies virus to produce a virus with reduced virulence for mice and teaches thymidine kinase deletions only, but does not suggest other attenuating deletions, nor does it suggest insertion of foreign DNA sequences. The other example involves the deletion of a small DNA sequence around a HindIII restriction site in the repeat region (16). From this work a patent application has been filed in Europe that involves other larger deletions in the unique short region as well. Published on May 15, 1985, European Patent Publication No. 0141458, based upon European Patent Application No. 84202474.8, filed on Oct. 12, 1984, entitled "Deletion Mutant of a Herpesvirus and Vaccine Containing Same", describes deletions in the unique short region of pseudorabies virus and their attenuating effect.
The present invention concerns deletions which have been introduced into the pseudorabies genome at sites previously undisclosed. These deletions are shown to be attenuating and to increase the utility of the virus as a vector for the expression of foreign genes as a vaccine. Foreign DNA sequences have been introduced into the attenuated pseudorabies virus and expressed as proteins. One embodiment of the invention concerns a vaccine useful for preventing pseudorabies and other swine diseases with a single inoculum.
Other relevant pseudorabies literature concerns the presence of naturally-occurring deletions in the genome of two vaccine strains of pseudorabies viruses (14, 17). These deletions are responsible, at least in part, for the attenuated nature of these vaccines. Such naturally-occurring deletions do not teach the methods for making these deletions starting with wild type pseudorabies virus DNA, nor do they suggest other locations at which to make attenuating deletions. Our deletions do not occur at the sites of these natural deletions, nor do they overlap these deletions in any way. Thus the presence of these naturally occurring deletions is simply a curious phenomenon that does not teach or instruct the current invention. There are no examples of naturally-occurring insertions of foreign DNA in herpesviruses.